Search Results (146)

Efferocytosis—the tightly regulated clearance of apoptotic cells by phagocytes—maintains tissue homeostasis and is impaired in chronic obstructive pulmonary disease (COPD), where it contributes to persistent inflammation and increases the risk of comorbidities, including lung cancer. Inhaled corticosteroids (ICS) and long-acting β2 agonists (LABAs) are cornerstones of COPD therapy, but their effects on efferocytosis and on the COPD–lung cancer interface are incompletely understood. The primary objective of this study was to determine whether the ICS fluticasone propionate and the LABA salmeterol xinafoate, alone or in combination at clinically informed concentrations (10⁻⁸–10⁻⁶ M; 10⁻⁴ M reserved for cytotoxicity screening), modulate efferocytic capacity and inflammatory cell survival across diverse phagocyte models. We performed standardized in vitro efferocytosis assays using murine peritoneal and alveolar macrophages, the murine macrophage line J774A.1, PMA-differentiated human THP-1 macrophages, human blood-derived neutrophils, and the human alveolar adenocarcinoma cell line A549. Apoptosis was induced in Jurkat T cells by UV irradiation (100 mJ/cm²) and in murine thymocytes by dexamethasone (1 µM, 4 h); apoptotic and necrotic populations were characterized by annexin-V/propidium iodide and Sytox Green/Hoechst H-33342 staining. Peritoneal macrophages showed the highest efferocytic activity (~75%), followed by J774A.1 (~75% at 24 h), THP-1 (~30% at 2 h; ~60% at 24 h), alveolar macrophages (~40%), and A549 cells (<20%). Neither fluticasone nor salmeterol, individually or in combination, significantly altered efferocytic capacity in any phagocyte tested (all ANOVA p > 0.26). Fluticasone (10⁻⁸ and 10⁻⁶ M) significantly improved 24 h neutrophil survival and reduced early apoptosis (p < 0.05) but did not translate this survival benefit into enhanced efferocytosis. Salmeterol was cytotoxic at 10⁻⁴ M and inactive at 10⁻⁸–10⁻⁶ M. These findings indicate that the established anti-inflammatory benefits of ICS/LABA in COPD do not extend to augmentation of efferocytosis in this acute, serum-free in vitro setting and that pharmacological restoration of efferocytosis in COPD—a defect implicated in the pathogenesis and progression of comorbid lung cancer—will likely require strategies targeting the efferocytic machinery itself (e.g., MerTK, Rac-1, MFG-E8) rather than relying on current inhaled therapy. Full article

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex, multi-system disease characterized by a multitude of symptoms across various organ systems. Diagnosis has relied heavily on heterogeneous clinical symptom presentation and evolving case definitions, with treatment focused on addressing presenting symptoms due to the paucity of validated biomarkers. Meanwhile, advances have been made in understanding the underlying pathophysiology through strong epidemiologic, clinical, and basic science studies. This narrative review synthesizes recent advances that are likely to drive a shift in understanding from symptom-based classification toward a molecularly defined understanding of the disease. This shift in understanding will likely provide the foundation for future research efforts focused on targeting diagnosis and treatment more effectively. Specifically, we reference the identification of rare genetic risk variants through the HEAL2 deep learning framework, the large-scale DecodeME genome-wide association study, and dynamic epigenetic markers of disease state. In addition, the findings revealed the downstream consequences of this genetic and epigenetic priming: chronic innate immune activation, CD8+ T cell exhaustion characterized by upregulation of the exhaustion-driving transcription factors Thymocyte Selection-Associated HMG Box (TOX) and Eomesodermin (EOMES), and a cellular energy crisis centered on mitochondrial dysfunction. Furthermore, results of recent studies have revealed sex-specific transcriptomic and proteomic signatures of maladaptive recovery. We also highlight the role of machine learning and artificial intelligence integrations in translating high-dimensional multi-omics data into actionable biological insights, including the identification of monocyte subsets via Positive Unlabeled Learning, circulating cell-free RNA diagnostic signatures, and integrated multi-modal disease models such as BioMapAI. The combination of these findings, which highlight multiple identifiable mechanisms of molecular activity, support the feasibility of molecular subtyping, precision diagnostics, and targeted therapeutic strategies for ME/CFS. Full article

Background: Chimerism analysis is a key tool for monitoring donor-cell engraftment and the risk of relapse and graft-versus-host disease (GVHD) following allogeneic hematopoietic cell transplantation (allo-HCT). The advantage of lineage-specific chimerism assessment, and its dynamics in patients receiving post-transplant cyclophosphamide (PTCy)-based GVHD prophylaxis, remains unclear. Objective: This review summarizes the current state of the art on chimerism analysis in patients with myeloid neoplasms undergoing allo-HCT with PTCy, with emphasis on lineage-specific testing and modern methodologies. Methods: A structured literature review was conducted to assess chimerism dynamics in whole blood (WB), bone marrow, and peripheral blood (PB) subpopulations, including T-cells, CD34⁺, myeloid, B, and NK (natural killer) cells, and their association with clinical outcomes following PTCy. Results: Lineage-specific PB chimerism, particularly in T-cells, myeloid lineage and CD34⁺ cells, is more sensitive than WB chimerism for predicting relapse. Declining donor myeloid chimerism or persistent myeloid mixed donor chimerism (MDC) may precede hematologic relapse and provide an early signal of graft instability or ineffective graft-versus-leukemia activity. T-cell MDC has been associated with an increased risk of relapse and a lower risk of GVHD, although persistent T-cell MDC in some patients may instead indicate immune tolerance. Declining CD34⁺ donor chimerism correlates with a higher risk of relapse and inferior survival outcomes and may therefore complement measurable residual disease testing. Data regarding B-cell and NK-cell chimerism remain inconsistent, likely influenced by delayed immune reconstitution. Compared to anti-thymocyte globulin, PTCy may promote higher donor T-cell chimerism, though findings across studies are variable. Next-generation sequencing (NGS) enables more sensitive detection of microchimerism and relapse prediction. Conclusions: Chimerism analysis, particularly when lineage-specific and NGS-based, offers valuable prognostic insight in allo-HCT with PTCy. Further prospective studies are needed to standardize testing and guide personalized post-HCT strategies. Full article

Background/Objectives: Allogeneic hematopoietic stem cell transplantation is associated with increased cardiovascular risk driven by endothelial dysfunction, chronic inflammation, and treatment-related metabolic disturbances, including dyslipidemia. In the contemporary era of post-transplant cyclophosphamide-based prophylaxis, the prognostic significance of dyslipidemia—particularly as assessed by non-HDL cholesterol—remains unclear. In this study, we aimed to compare the engraftment days, graft-versus-host disease (GVHD) development, relapse, overall survival rates, and cardiovascular mortality in patients using myeloablative/reduced intensity conditioning regimens with post-transplant cyclophosphamide (PTCy) 50 mg/kg/day for 2 days in patients with acute leukemias. Methods: A total of 95 adult patients with acute leukemias were included in their first remission who underwent matched sibling donor transplantation with PTCy (50 mg/kg on days +3 and +4). Patients were stratified according to pre-transplant non-HDL-C levels (<160 mg/dL vs. ≥160 mg/dL). Matched related donors were selected for the patients. All patients received either myeloablative or reduced-intensity conditioning based on EBMT criteria, with fludarabine-based combinations including busulfan, treosulfan, or TBI, along with ATLG administered at a total dose of 15 mg/kg. Peripheral blood stem cells were used for all transplants, and GVHD prophylaxis consisted of cyclosporine. Results: Platelet (median 13 vs. 14 days) and neutrophil (median 14 vs. 15 days) engraftment times and veno-occlusive disease (VOD) rates were comparable across groups (all p > 0.05); cumulative incidences of grade II–IV aGVHD at +100 days, grade III–IV aGVHD at +100 days, and moderate-severe cGVHD at 1 year, relapse-free survival, and non-relapse mortality at 1 year were comparable in two cohorts (all p > 0.05). GVHD-free/relapse-free survival (GRFS) at 1 year was also comparable across groups (p = 0.15). Median GRFS was 150 (95% CI: 120–330) days and 270 (95% CI: 154-not reached) days, respectively [HR was 0.68 (0.40–1.15), p = 0.15; GRFS at 1 year was 66.6% vs. 52.0%, respectively]. The groups were also comparable in terms of overall survival (OS). Follow-up ranged from 0.5 to 108 months, and median follow-up was 60 months in two cohorts. Median OS was not reached in non-HDL-C < 160 (95% CI: 70 months–not reached) and 67 months in non-HDL-C ≥ 160 groups (95% CI: 13 months–not reached) (Log rank = 0.21). No cardiovascular death events occurred during the follow-up period. Conclusions: In this homogeneous matched sibling donor transplant cohort with extended follow-up and uniform administration of post-transplant cyclophosphamide, cyclosporine-based GVHD prophylaxis, and anti-thymocyte lymphoglobulin (ATLG), pre-existing dyslipidemia was not associated with an adverse impact on GRFS, NRM, PFS, CMV reactivation, OS or long-term cardiovascular mortality. Full article

T-cell lymphoblastic lymphoma (T-LBL) is an aggressive malignancy of immature T-cells accounting for a substantial proportion of pediatric non-Hodgkin lymphoma cases. Current chemotherapeutic regimens achieve five-year event-free survival rates of 80–90%, yet relapse occurs in approximately 20% of patients and remains a major therapeutic challenge. This underscores the need for improved, molecularly informed treatment strategies. Recent genomic profiling has highlighted the central role of NOTCH1 signaling in T-LBL pathogenesis. NOTCH1, a transmembrane receptor critical for T-cell differentiation and maturation, requires tightly regulated activation during normal thymocyte development. Dysregulated signaling disrupts this balance, driving aberrant proliferation and impaired differentiation, characteristics of malignant transformation. While activating mutations have long been recognized as key oncogenic events, the recent identification of recurrent NOTCH1 translocations, associated with adverse outcomes, reveals an additional mechanism of pathway activation. These findings reinforce NOTCH1 as a pivotal oncogenic hub in T-cell malignancies and a compelling target for therapeutic intervention. This review synthesizes current insights into the molecular landscape of pediatric T-LBL, with a focus on the biological and clinical implications of NOTCH1 mutations and translocations. Furthermore, we examine emerging approaches to therapeutically exploit aberrant NOTCH1 signaling for the more precise and effective treatment of this disease and formulate outstanding research questions. Full article

Age-related thymic atrophy, a hallmark of immunosenescence linked to age-related diseases, involves gonadal and adrenal steroid hormones, but their distinct roles and mechanisms in this process remain unclear. Through biochemical, histological, and RNA-seq analyses, we comprehensively explored the mechanisms underpinning age-related thymic atrophy in response to ovariectomy (OVX) versus adrenalectomy (ADX) in female mice. Compared to the sham controls, OVX overtly ameliorated age-related thymic atrophy, as evidenced by increased thymus mass, a larger gross thymus area, and denser cortex cellularity. In contrast, ADX evidently accelerated age-related thymic atrophy, characterized by increased adipose infiltration and decreased cortex/medulla ratio, obscure cortico-medullary junctions, and sparser thymic cortical cells. Unexpectedly, combined OVX and ADX displayed a more pronounced effect than OVX alone in ameliorating age-related thymic atrophy. Mechanistically, OVX decreased while ADX increased the circulating 17β-estradiol levels in female mice, which drove these opposing outcomes potentially by promoting Pparg-mediated thymic fat deposition and blocking Cdk1-dependent thymocyte cell cycle progression. Although OVX eliminated gonadal 17β-estradiol production, it appeared to trigger a compensatory adrenal-dependent estrogen biosynthesis, whereas combined OVX and ADX nearly eliminated all estrogen sources, thus leading to a more pronounced effect than OVX alone in ameliorating age-related thymic atrophy in female mice. Notably, OVX increased while ADX decreased serum corticosterone levels, but these alterations exerted minimal impacts on age-related thymic atrophy, highlighting a pivotal role of estrogens over glucocorticoids in accelerating age-related thymic atrophy in females. Undesirably, although OVX ameliorated age-related thymic atrophy, it appeared to simultaneously increase autoimmune susceptibility by downregulating thymic Cd74 expression. Taken together, our results indicate that OVX ameliorates while ADX accelerates age-related thymic atrophy in females. Estrogens rather than glucocorticoids act as the predominant regulator of this process, potentially via promoting Pparg-dependent fat deposition and blocking Cdk1-dependent thymocyte cycle progression. However, OVX-induced estrogen depletion also elevated autoimmune risk, emphasizing the need to balance benefits and risks in regulating thymic aging. Full article

The thymus, as the primary lymphoid organ for T cell development, orchestrates a complex continuum of processes encompassing precursor migration, lymphocyte lineage commitment, and antigen-guided selection to generate a self-tolerant and immunocompetent T cell repertoire. The thymus is anatomically divided into the cortex, which facilitates the positive selection of thymocytes through interactions between T cell receptors and self-peptide–MHC complexes on cortical epithelial cells, and the medulla, which mediates negative selection by medullary epithelial cells in concert with dendritic cells via the presentation of self-antigens. Key regulatory elements controlling thymocyte development include the transcription factors ThPOK/Runx3 and Sox13/PLZF, chemokine-driven migration mediated by CXCR4 and CCR7, and cytokine signaling. These components collectively exert a profound influence on the final outcome: the establishment of TCR affinity thresholds for tissue-specific antigens in mature T cells. In summary, the integration of multidimensional methodologies highlights the pivotal role of the thymus in immune tolerance, with translational implications for autoimmunity, cancer immunotherapy, and regenerative medicine, as reviewed herein. Full article

Background: T cell regeneration in the thymus is intrinsically linked to the T cell-biased lineage differentiation of hematopoietic stem and progenitor cells (HSPCs). Although nonhuman primates (NHPs) serve as indispensable models for studying thymic output under physiological and pathological conditions, a non-animal technology facilitating efficient TCR-selected T cell development and evaluating T cell output from NHP-derived HSPCs has been lacking. To address this gap, we established a rhesus macaque-specific artificial thymic organoid (RhATO) modeling primary thymus-tissue-free thymopoiesis. Methods: The RhATO was developed by expressing Rhesus macaque (RM) Delta-like Notch ligand 1 in mouse bone marrow stromal cell line (MS5-RhDLL1). The bone marrow-derived HSPCs were aggregated with MS5-RhDLL1 and cultured forming 3D artificial thymic organoids. These organoids were maintained under defined cytokine conditions to support complete T cell developmental ontogeny. T cell developmental progression was assessed by flow cytometry, and TCR-selected subsets were analyzed for phenotypic and functional properties. Results: RhATOs recapitulated the complete spectrum of thymopoietic events, including emergence of thymus-seeding progenitors, CD4⁺CD3⁻ immature single-positive and CD4⁺CD8⁺ double-positive early thymocytes, and mature CD4⁺ or CD8⁺ single-positive subsets. These subsets expressed CD38, consistent with the recent thymic emigrant phenotype, and closely mirrored canonical T cell ontogeny described in humans. RhATO-derived T cells were TCR-selected and demonstrated cytokine expression upon stimulation. Conclusions: This study provides the first demonstration of an NHP-specific artificial thymic technology that faithfully models thymopoiesis. RhATO represents a versatile ex vivo platform for studying T cell development, immunopathogenesis, and generating TCR selected T cells. Full article

Introduction: Simultaneous liver–kidney (SLK) transplant recipients are considered at lower immunologic risk than kidney-alone recipients, so steroid-only induction is often used. However, some centers continue to include basiliximab induction in their protocols. This study compared graft and infectious outcomes in SLK recipients receiving basiliximab (Bas) induction versus those without basiliximab (No Bas). Methods: Using TriNetX, we conducted a retrospective, propensity-score-matched study of SLK recipients comparing 3-, 6-, and 12-month graft and infectious outcomes. Patients receiving alemtuzumab or anti-thymocyte globulin were excluded; steroid induction was permitted but not required in either cohort. Maintenance immunosuppression included tacrolimus, mycophenolate, and prednisone. Cohorts were matched on 71 variables, including demographics, disease etiology, severity markers, and cPRA. Results: After matching, 292 patients were included per cohort (mean age 56.9 ± 10.1 years; 61% male). Kidney and liver rejection rates were similar. The No Bas cohort had more liver biopsies (25.5% vs. 18.2% at 1 year, p = 0.04). Kidney biopsy, graft failure, re-transplantation, delayed graft function, and mortality were comparable. Liver primary non-function was more frequent in Bas (2.8% vs. 0.4%, p = 0.04). The No Bas cohort had higher CMV at 3 months (13.4% vs. 6.7%, p = 0.008) and higher EBV at all time points (4.0% vs. 0.4% at 1 year, p = 0.004). Conclusions: SLK recipients without basiliximab induction had comparable rejection outcomes but more viral infections, potentially from greater steroid exposure, and more liver biopsies, which may reflect higher clinical suspicion for rejection or incomplete capture of rejection events in EMR data. Full article

Background: Graft-versus-host disease (GVHD) is a rare but serious complication following solid organ transplantation (SOT), particularly in transplants involving organs with a high volume of passenger donor T-lymphocytes. This case highlights the clinical course and diagnostic challenges of GVHD following simultaneous pancreas and pre-emptive kidney transplantation. Methods: A 51-year-old male with long-standing type 1 diabetes mellitus underwent simultaneous pancreas and kidney transplantation with induction therapy using rabbit anti-thymocyte globulin and methylprednisolone. Three months post-transplant, he presented with a diffuse lichenoid cutaneous eruption. Diagnostic evaluation included an extensive infectious workup, skin punch biopsy, liver and bone marrow biopsies, and microchimerism assay. Results: Skin biopsy revealed interface vacuolar dermatitis consistent with cutaneous GVHD. Subsequent liver and bone marrow biopsies confirmed GVHD involvement, with microchimerism assay showing 43% donor-origin T-cells in the bone marrow. Initial treatment with systemic and topical corticosteroids led to temporary improvement. However, the patient developed bone marrow suppression, recurrent bacteremia, and invasive fungal infection, resulting in a prolonged ICU stay and ultimately death. Conclusions: This case underscores the importance of considering SOT-GVHD in patients receiving organs rich in donor lymphocytes, such as pancreas transplants. Early recognition and multidisciplinary management are critical to improving outcomes in this rare but life-threatening condition. Full article

Background/Objectives: Heart transplantation (HTx) is a lifesaving procedure for end-stage heart failure patients; however, postoperative infections remain a major challenge due to immunosuppressive therapy and surgical complications. Traditional biomarkers such as C-reactive protein (CRP) and procalcitonin (PCT) have limitations in distinguishing infections from systemic inflammatory response syndrome (SIRS). Emerging markers such as Presepsin and interleukin-6 (IL-6) may improve diagnostic accuracy. This study aimed to evaluate the kinetics and reliability of these four inflammatory biomarkers in heart transplant recipients in the immediate postoperative period. Methods: This retrospective observational study included 126 patients who underwent HTx at Policlinic of Bari between January 2022 and November 2024. Patients were categorized into infected (n = 26) and non-infected (n = 100) groups based on clinical and microbiological criteria. Biomarkers (CRP, PCT, Presepsin, and IL-6) were measured preoperatively and on postoperative days (PODs) 1, 2, 3, 4, 5, and 10. Statistical analyses included the Mann–Whitney U test and logistic regression to identify the independent predictors of infection. Results: CRP and PCT levels differed significantly between the groups only on day 10, limiting their use as early infection markers. In contrast, Presepsin levels were significantly elevated in infected patients from day 1 (p < 0.001), whereas IL-6 levels showed significant differences from day 3 onward. Presepsin showed the strongest association with infection in the early postoperative phase. Conclusions: Presepsin and IL-6 outperformed CRP and PCT in detecting early postoperative infections in heart transplant recipients. Their early elevation supports their use as reliable markers for guiding timely clinical intervention and improving patient outcomes. Further research is needed to validate these findings in larger cohorts and with different immunosuppressive regimens. Full article

Designed hen eggs enriched in DHA and EPA are an alternative source of essential phospholipids. This study assessed the effects of ovophospholipids on lymphocyte subsets in non-immunized mice and on the humoral immune response in sheep red blood cells (SRBC)-immunized mice. Ovophospholipids were administered orally for 14 days (once a day) at doses of 100, 10, and 1 mg/kg. Ovophospholipids increased the total lymphocyte count of in the lymphoid organs. At 10 and 1 mg/kg, ovophospholipids increased the subsets of CD4⁻CD8⁻ and CD4⁺CD8⁺ thymocytes but decreased the percentage of CD4⁺ and CD8⁺ thymocytes. A stimulating effect on splenocytes was particularly evident 24 h after administration of the 10 and 1 mg/kg doses. Ovophospholipids also elevated the absolute counts of CD3⁺ and CD19⁺ splenocytes. An increase in the absolute count of CD3⁺, CD4⁺, CD8⁺, and CD19⁺ lymphocytes of the mesenteric lymph nodes was observed 24 h after administration of the lowest dose. The increase in the percentage and absolute count of CD19⁺ cells and in the absolute count of CD3⁺ cells was still observed after 72 h. At all doses, ovophospholipids elevated the number of plaque-forming cells on day 4 and increased 2-mercaptoethanol-resistant antibody titer on day 7 after priming. In conclusion, ovophospholipids can modulate the immune response in mice. Full article

Effective immunosuppressant pharmacotherapy is essential for successful organ transplantation. Background/Objectives: Generally, induction therapy includes basiliximab (BAS) or anti-thymocyte globulin (THY). However, other biological molecules have been used to accelerate firm immunosuppression. A reduced effectiveness of these induction agents increases the risk of graft rejection. This study aims to evaluate the ineffectiveness rate of biological molecules based on spontaneous reports uploaded to the EudraVigilance database. Methods: Specific topics related to the safety profiles of alemtuzumab, BAS, belatacept, and THY were analyzed. A total of 23 preferred terms describing drug resistance, drug ineffectiveness, and transplant rejection were used as the inclusion criteria. Descriptive and disproportionality analyses were performed. Results: Regarding the four molecules, 18,564 safety reports were communicated, with n = 5089 (27.4%) for THY and n = 3469 (18.7%) for BAS. Most adverse drug reactions (ADRs) for THY, BAS, and belatacept affected the adult male population. As expected, the majority of the ADRs were linked to infections, followed by general disorders. BAS presented higher probabilities of drug resistance and transplant rejection being reported among the four molecules. A higher probability of reporting drug ineffectiveness was noted for THY than for the other molecules. Conclusions: All the molecules showed small frequencies regarding resistance. As expected, transplant rejection was more frequently reported for all molecules (especially for BAS), accompanied by a notable variability in reporting frequencies. However, a causal relationship between the reported adverse reactions and drug efficacy cannot be established based on the present results. Further real-world evidence studies will enhance our understanding of the safety and efficacy of these drugs in transplant patients. Full article

Certain strains of Influenza A virus (IAV), a primary cause of influenza, can lead to pneumonia. Patients recovering from influenza pneumonia may experience physical discomfort akin to post-acute sequelae of COVID-19 (PASC). Despite extensive clinical research on viral pneumonia during convalescence, animal model studies are scarce, highlighting the need for a reliable model for pharmaceutical research. In this study, BALB/c mice were divided into three groups: NC (control), MC (infected with IAV), and Model (treated with oseltamivir post-infection for five days). A fatigue model was then induced in the Model group through diet restriction and weight-bearing swimming. The results showed the MC group had a 75% survival rate, while the NC and Model groups had 100%. Both the MC and Model groups experienced rapid weight loss followed by gradual recovery, differing significantly from the NC group. From dpi (days post-inoculation) 6 to dpi9, the MC group lost more weight than the NC group. The MC group had the highest pulmonary index, but there was no significant difference in IAV Nucleoprotein (NP) expression across groups. The Model group had higher IL-10 levels than the NC and MC groups, while the MC group had the highest TNF-α expression. Hematoxylin and eosin (H&E) staining revealed pathological changes in the MC and Model groups, with severe damage and pulmonary fibrosis in the MC group. Oxidative stress markers showed the MC group had the highest lactate dehydrogenase (LDH) and malondialdehyde (MDA) levels and lowest superoxide dismutase (SOD) activity. Electron microscopy indicated mitochondrial damage in both the MC and Model groups. The Model group had the lowest splenic and thymic indices, with histological findings showing larger splenic nodules in the MC group and poor thymocyte density and atrophy in the Model group. The successful creation of this mouse model of influenza pneumonia convalescence phase fatigue, exhibiting fatigue syndrome with various symptoms, holds significance for PASC and other viral pneumonia convalescence phase animal model research. Full article

Background: The thymus is the central hub of T-cell differentiation, where epithelial cells guide the process of their maturation. Objective: Our goal was to identify and describe progenitor cells within the human thymus that can differentiate into epithelial cells. Methods: When we plated enriched thymic cells in 3D culture conditions, rare individual cells capable of self-renewal and differentiation formed spheroids. Results: Both neonatal and adult thymuses produced similar numbers of spheroids, suggesting that progenitor potential remains consistent across age groups. Some cells within the spheres express genes typical of mature epithelial cells, while others express genes associated with the immature compartment active during thymic organogenesis. However, there were also cells expressing PDGFRβ. We treated the tissues with 2-deoxyguanosine before digestion, which improved the yield of progenitor cells. We also cultured the enriched stromal thymocytes with Cyr61 and Interleukin-22, which affected the spheroid size. Conclusions: Our efforts towards thymic reconstitution are ongoing, but our research uncovers previously unknown characteristics of the elusive epithelial progenitor population. Full article